Lipids play essential roles in all living cells, not only as key components of membranes and as a source of energy, but also as important mediators of cellular signaling. At the organismal level, individual differences in lipid metabolism can impact a variety of human diseases. In our laboratory, ethylnitrosurea (ENU) mutagenized zebrafish are screened in vivo for abnormality in lipid processing using the fluorescent reporters. One of the lethal recessive mutant, fat-free, was identified at the beginning of the large-scale screen. Biochemical analysis using fluorescent lipids analogues indicate that fat-free mutation may attenuate biliary synthesis or secretion. Although three genes (FIC1, BSEP, and MDR3) responsible for infants and children with progressive familial intrahepatic cholestasis (PFIC) are identified, it remains unknown if there are undiscovered genes that are involved in bile synthesis and/or regulation may lead to PFIC. Fat-free locus is not relevant to FIC1, BSEP or MDR3 because all three genes are mapped to different linkage groups using zebrafish radiation hybrid panel. Therefore, fat-free mutant provides an ideal potential model to discover a gene that may contribute to PFIC. The specific aims of this proposal are positionatly clone and characterization of the fat-free gene. The PCR-based analysis methods have enabled me to locate a region that is 0.06 cM (1 out of 1700 meioses) from the fat-free locus. The final stage of mapping is relied on BAC walking, SNP detection, and shotgun sequencing. To characterize fat-free gene, morpholino knock-down, genomic DNA rescue, whole mount in situ hybridization, and microarray analysis are included.